HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other’s transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions.


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The RNA sequencing which is listed in Supplementary file 1-3 are deposited in Gene expression omnibus data base and the GEO accession number for data generated for this paper is GSE190212. The source data for all the figures will be submitted prior to the publication.
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Sample sizes for cell based assays were chosen according to the standards in the field or by previously published studies that have similar methods (at least two to three independent biological replicates for each condition) which gave sufficient statistical power for the effect sizes of interest. They were not predetermined based upon statistical methods given the inability to predict effect size for the cell experiments No data were excluded from the analysis All the gene expression analysis, RIPs, ChOPs etc and functional assays such as cell proliferation assays, apoptosis and cell cycle analysis were performed using 2 to 3 biological replicates and the data in all replicates were consistently reproducible. All the immunoblots were replicated in independent experiments and the replicate immunoblots showed the similar results. RNA sequencing for knockdown samples and FGF-2 treated samples were performed in two biological replicates. Sample correlation cluster analysis was used to assess successful reproducibility. All experiments were consistently reproducible. All graphs include individual data points from biological replicates. The mice were randomly allocated into the cages before the treatment. The 4-6 weeks old male and female mice were allocated randomly into two groups (control sh group and IER3-AS1 sh group). All experiments were performed with appropriate negative and positive controls in keeping with the standards of the field.

March 2021
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